Methionine is the universal starting amino acid residue for growing peptide chains (e.g., protein synthesis) in most living systems. In order for methionine to function as the initiating amino acid for nascent peptide synthesis in bacteria, it must undergo a series of transformations both before initiation of protein synthesis and after the protein has been made (see review by Meinnel, T., Mechulam, Y., and Blanquet, S., 1993). In E. Coli, all of the enzymes involved in these transformations and their respective genes have been isolated and/or sequenced (Meinnel, T. and Blanquet, S., 1994).
Two of the most notable transformations in the initiating amino acid methionine amino acid involve the addition of an N-formyl group to methionine molecules prior to the initiation of messenger RNA (mRNA) translation and the subsequent removal of the N-formyl group from the amino (N-) terminal methionine of the nascent peptide. The removal of the N-formyl group is accomplished by the enzyme peptide deformylase (EC 3.5.1.27 according to the IUB nomenclature as published in "Enzyme Nomenclature Recommendations" (1992) Academic Press, San Diego). Peptide deformylase (PDF) cleaves the formyl group from most nascent formyl-methionine-peptides in a substrate specific reaction. However, there are exceptions to the routine action of PDF. For example, some E. coli proteins remain either wholly or partially formylated. (Hauschild-Rogat, P., 1968; Marasco, W. A., et al., 1984; and Milligan, D. L. and Koshland, Jr., D. E., 1990). It has also been observed that several recombinant proteins which are normally free of N-formyl-methionine (f-Met) exhibit a significant retention of f-Met when they are overproduced in recombinant E. coli strains. Examples of this phenomenon include E. coli tryptophan synthase alpha- and beta-subunits (Sugino, Y., et al., 1980; Tsunasawa, S., et al., 1983); bovine somatotropin (BST) (Bogosian, G., et al., 1989); eel growth hormone (Sugimoto, Y., et al., 1990); E. coli 1-acyl-sn-glycerol-3-phosphate acyltransferase (Coleman, J., 1992); human granulocyte colony-stimulating factor (Clogston, C. L., et al., 1992); bovine fatty acid-binding protein (Specht, B., et al., 1994); bovine cytochrome P450 (Dong, M. S., et al. 1995); methanothermus fervidus histone A (Sandman, K., et al., 1995); human interleukin-5 (Rose, K., et al., 1992); human parathyroid hormone (Rabbani, S., et al., 1988; Hogset, A., et al., 1990); human gamma-interferon (Honda, S., et al., 1989); E. coli threonine deaminase (Eisenstein, 1991); and E. coli To1Q membrane protein (Vianney, A., et al., 1994).
Retention of the formyl group on a protein expressed in and purified from bacterial expression systems is undesireable when preparing recombinant pharmaceuticals. As a result, complex and expensive purification procedures are required to purify the deformylated protein of interest to a degree sufficient to qualify it for pharmaceutical use. In addition, costly analytical methods to quantify the formylated isoform must frequently be devised in order to insure that the level of such isoform in the final product is below a desired level. Therefore, there exists a need to achieve the efficient removal of undesirably retained N-formyl groups on recombinant proteins without interfering with the level of recombinant protein production in bacterial expression systems.